Use of Quaternary Pyridinium Salts as Vasoprotective Agents

ABSTRACT

The invention relates to the use of quaternary pyridinium salts of formula (I), 
     
       
         
         
             
             
         
       
     
     wherein R is NH 2 , CH 3 , or N(H)CH 2 OH group, and X is pharmaceutically acceptable counterion, for the preparation of vasoprotective agent for the treatment or prevention of conditions or diseases associated with dysfunction of vascular endothelium, oxidative stress, and/or insufficient production of endothelial prostacyclin PGI 2 , in particular but not exclusively if the above coincides with hypercholesterolemia, hypertriglyceridemia or low HDL level.

The present invention relates to the use of certain quaternarypyridinium salts for the preparation of a vasoprotective agent for thetreatment and/or prevention of conditions or diseases associated withdysfunction of vascular endothelium, oxidative stress, and/orinsufficient production of endothelial prostacyclin (PGI2), inparticular but not exclusively if the above coincides withhypercholesterolemia/hypertriglyceridemia, as well as the use ofpyridinium salts for oral use in diet supplementation.

There is increasing evidence that endothelial dysfunction plays a keyrole in the formation and progression of atherosclerotic plaque.Endothelial dysfunction has recently gained diagnostic, prognostic andtherapeutic significance in atherothrombosis (Heitzer T, Schlinzig T,Krohn K, Meinertz T, Munzel T. Endothelial dysfunction, oxidativestress, and risk of cardiovascular events in patients with coronaryartery disease. Circulation 2001;104:2673-2678; Schachinger V, Britten MB, Zeiher A M. Prognostic impact of coronary vasodilator dysfunction onadverse long-term outcome of coronary heart disease. Circulation2000;101:1899-1906; Perticone F, Ceravolo R, Pujia A, Ventura G,Iacopino S, Scozzafava A, Ferraro A, Chello M, Mastroroberto P,Verdecchia P, Schillaci G. Prognostic significance of endothelialdysfunction in hypertensive patients. Circulation 2001;104:191-196;Suwaidi J A, Hamasaki S, Higano S T, Nishimura R A, Holmes D R, Jr.,Lerman A. Long-term follow-up of patients with mild coronary arterydisease and endothelial dysfunction. Circulation 2000;101:948-954).Clinically, endothelial dysfunction is identified as impairement ofbiological activity of NO, which is diagnosed as an impairment ofvasodilating NO activity. Impairment of NO activity coincides withoxidant stress (Heitzer T, Schlinzig T, Krohn K, Meinertz T, Munzel T.Endothelial dysfunction, oxidative stress, and risk of cardiovascularevents in patients with coronary artery disease. Circulation2001;104:2673-2678) and impairment of PGI2 synthesis (Kyrle P A, MinarE, Brenner B, Eichler H G, Heistinger M, Marosi L, Lechner K.Thromboxane A₂ and prostacyclin generation in the microvasculature ofpatients with atherosclerosis—effect of low-dose aspirin. Thromb Haemost1989;61:374-377), although systemic level of PGI₂ may be elevated.Indeed, several years ago it was proposed that increased lipidperoxidation might promote development of atherosclerosis owing toselective impairment of prostacyclin synthesis in endothelial cells andsubsequent activation of platelets (Gryglewski R J. Prostacyclin andatherosclerosis. TIPS 1980;1:164-1.68; Gryglewski R J. Prostaglandins,platelets, and atherosclerosis. CRC Crit Rev Biochem 1980;7:291-338;Gryglewski R J, Szczeklik A. Prostacyclin andatherosclerosis—experimental and clinical approach. 1983;213-226). Thisconcept was then supported experimentally. It is apparent now thatimpairment of PGI2 synthesis in endothelium may lead to the excessivestimulation of TP receptors in endothelium and vascular smooth musclecells by TXA₂, PGH2 or other eicosanoids, and to subsequentvasoconstriction, platelet aggregation, and inflammatory response ofendothelium as well as endothelial apoptosis (Chlopicki S, Gryglewski RJ. Endothelial secretory function and atherothrombosis in “TheEicosanoids”, chapter 23, 267-276. ed. P. Curtis-Prior, John Wiley andSons, Ltd, 2004). This means that deficiency of PGI₂ may trigger orenNance inflammatory and thrombotic processes in vascular wall, whichare now considered to be the key elements of atherosclerosis.

It has been accepted that elevated low-density lipoprotein cholesterol(LDL) and/or triglyceride (TG) plasma levels represent major riskfactors for the development of atherosclerosis (Levine G N, Keaney J FJr, Vita J A. Cholesterol reduction in cardiovascular disease. Clinicalbenefits and possible mechanisms. N Engl J Med 1995;332:512-521).Moreover, a low plasma level of high-density lipoprotein cholesterol(HDL) is an important independent risk factor of atherosclerosis. HDLhas the potential ability to prevent and correct endothelial dysfunctionby increasing availability of NO and PGI₂. (Ng DS. Treating low HDL—frombench to bedside. Clin Biochem 2004;37:649-659; Chapman M J, Assman G,Fruchart J C, Shepherd J, Sirtoti C. Raising high-density lipoproteincholesterol with reduction of cardiovascular risk: the role of nicotinicacid—a position paper developed by the European Consensus Panel onHDL-C. Curr Med Res Opin 2004;20:1253-1268; Calabresi L, Gomarashi M,Franceschini G. Endothelial protection by high-density lipoproteins.Arterioscler Thromb Vasc Biol 2003;290:2292-2300).

In WO00/40559 therapeutic and cosmetic uses of certain nicotinamidederivatives, 1,3-disubstituted pyridinium salts, including1-methylnicotinamide (MNA⁺) and 1-methyl-N′-(hydroxymethyl)nicotinamide(MNAF⁺) salts were disclosed. It was reported that said derivatives havethe utility in topical treatment of skin diseases, in particular cruralulceration, acne, psoriasis, atopic dermatitis, vitiligo, as well asburns and scalds and in wound healing. Said derivatives have also theactivity of promoting hair re-growth, therefore they are useful in thetreatment of hair loss of different origin. Different types of topicalformulations for administration of these compounds on the skin ormucosal surface are described, like shampoo, ointments, creams, gels,lotions, solutions, aerosols, etc., and for oral administration in thetreatment of skin diseases. Also, cosmetic action of these compounds wasdescribed, in particular regenerating and smoothing of the skin.

Effects of 1-methylnicotinamide chloride (MNA⁺) in some skin diseaseswere described in a recent publication (G

bicki J, Sysa-J

drzejowska A, Adamus J, Woźniacka A, Rybak M, Zielonka J.1-Methylnicotinamide: a potent anti-inflammatory agent of vitaminorigin. Pol J Pharmacol 2003;55:109-112). It has been proposed that MNA⁺displays anti-inflammatory action, though the mechanism of this effectwas not elucidated.

1-Methyl-3-acetylpyridinium salt (MAP⁺), was described in a publication(Takashi Sakurai, Haruo Hosoya. Charge transfer complexes ofnicotinamide-adenine dinucleotide analogs and flavine mononucleotide.Biochim. Biophys. Acta 1966;112(3):359-468).

Now it has been found that MAP⁺ and some of the compounds described inWO00/40559, in particular MNA⁺ and MNAF⁺ possess unique pharmacologicalproperties associated with their ability to release endogenousprostacyclin (PGI2) from vascular endothelium, which propertydistinguishes them from closely structurally related nicotinamide,nicotinic acid, trigonelline and endogenous MNA⁺ metabolites, such as1-methyl-2-pyridone-5-carboxyamide (2-PYR) and1-methyl-4-pyridone-3-carboxyamide (4-PYR). Surprisingly, as found bythe present inventors, certain compounds are endowed with the ability tocorrect the lipoprotein profile, in particular to lower LDL and/or TGplasma level and to raise HDL plasma level, leading toanti-atherosclerotic effects. Independently of the effect of certainquaternary pyridinium salts of the invention on lipoprotein profile,augmentation of PGI₂ synthesis by quaternary pyridinium salts found bythe present inventors may show therapeutic potential in many diseases asdiscussed below, where endothelial dysfunction, oxidant stress, and/orPGI₂ deficiency play a pathogenetic role, including those associatedwith hypercholesterolemia/hypertriglyceridemia.

BRIEF DESCRIPTION OF FIGURES OF THE DRAWING

FIG. 1 Scheme of the method for detection of thrombolytic action ofdrugs in vivo in rats (according to Gryglewski)

FIG. 2 Thrombolytic response induced by intravenous administration ofMNA⁺ in vivo (30 mg/kg)

FIG. 3 Changes in plasma levels of 6-keto-PGF_(1α)() and TXB₂ (∘) afterintravenous administration of MNA⁺ (30 mg/kg)

FIG. 4 Lack of thrombolytic response after intravenous administration ofnicotinamide or nicotinic acid (30 mg/kg).

FIG. 5 Lack of thrombolytic response after intravenous administration of2-PYR or trigonelline (30 mg/kg).

FIG. 6 Thrombolytic response induced by intravenous administration ofMAP⁺ in vivo (30 mg/kg)

FIG. 7 Changes in plasma levels of 6-keto-PGF_(1α)() and TXB₂ (∘) afterintravenous administration of MAP in vivo (30 mg/kg)

FIG. 8 Thrombolytic response induced by intravenous administration ofMNAF⁺ (30 mg/kg)

FIG. 9 Lack of effect of MNA⁺ on collagen-induced aggregation ofplatelets (1 mg/ml)

FIG. 10 Lack of effect of MNA⁺ on latex-induced activation ofneutrophils

The present invention provides in its first aspect the use of quaternarypyridinium salts of the formula I:

wherein R is NH₂, CH₃, or N(H)CH₂OH group, and X is pharmaceuticallyacceptable counterion,

for the preparation of vasoprotective agent for the treatment orprevention of conditions or diseases associated with dysfunction ofvascular endothelium, oxidative stress, and/or insufficient productionof endothelial prostacyclin PGI₂.

Preferably, said dysfunction of vascular endothelium, oxidative stress,and/or insufficient production of endothelial prostacyclin PGI₂ isassociated with hypercholesterolemia, hypertriglyceridemia or low HDLlevel.

Particularly advantageous activity of the compounds of the formula I istheir endothelial action associated with release of PGI₂, due to whichsaid compounds of formula I may improve perfusion in tissues, exertanti-aggregatory, thrombolytic, anti-apoptotic, anti-atheroscleroticactivity, and protect gastrointestinal mucosa.

The advantage of the invention is that thrombolytic action of thecornpounds of the formula I is not accompanied by hypotensive activity.

In one embodiment of the invention, said condition or the disease isatherosclerosis of vascular bed of any kind, including chronic coronarydisease, ischemic cerebrovascular episode or artherosclerosis of theextremities, including thromboangigitis obliterans.

In another embodiment said condition or the disease is acutecardiovascular event associated with atherosclerosis, in particularsudden cardiac death, acute coronary syndrome (including unstablecoronary artery disease, myocardial infarct), the necessity of coronaryangioplasty (PCI), coronary-aortal by-pass surgery (CABG), ischemicstroke, or peripheral circulation revascularization.

In yet another embodiment said condition or disease is selected from thegroup of risk factors for atherosclerosis, comprising the following:hypercholesterolemia, arterial hypertension, smoking,hyperhomocysteinaemia, insulin resistance, diabetes, menopause, aging,obesity, mental stress, infections, inflammatory states, includingperiodontal diseases, reumathoid arthritis, allograft vasculopathy ornitrate tolerance.

In yet another embodiment said condition or the disease is dyslipidemia;in particular hypercholesterolemia or hypertriglyceridemia in particularassociated with low plasma level of HDL.

In yet another embodiment said condition or disease is thrombosis thatis not related directly with atherosclerosis, in particular thrombosisassociated with implantation of metallic vascular prostheses (stents),coronary-aortal by-pass surgery (CABG), any type of surgery withextracorporeal circulation, hemodialysis, venous thrombo-embolicdisease.

In further embodiment said condition or disease is selected from thefollowing group: chronic heart failure, pulmonary hypertension,microvascular diabetic complications, like diabetic retinopathy andnephropathy, diabetic neuropathy, nephrotic syndrome, chronic renalfailure, adults respiratory distress syndrome (ARDS), cystic fibrosis,chronic obstructive pulmonary disease (COPD), preeclampsia/eclampsia,erectile dysfunction, Stein-Leventhal syndrome, sleep apnea, systemiclupus erythematosus, sickle cell anemia, non-specific inflammatory boweldiseases, gastric or duodenal ulcers, glaucoma, chronic liver disease,primary amyloidosis, neurodegenerative diseases, in particularneurodegenerative disease selected from vascular dementia, Alzheimer'sdisease and Parkinson's disease.

Also advantageous is the use of the compounds of formula I for thepreparation of a medicament for prophylaxis or treatment of gastric orduodenal ulcer.

In a further preferred embodiment said condition or disease is chronicliver disease, in particular chronic viral hepatitis.

In a further preferred embodiment said condition or disease is chronicobstructive pulmonary disease (COPD).

Said medicament may be in a form suitable for any administration route,such as oral, parenteral, intranasal or inhalation route. Such route ofadministration will depend of course on the particular state or diseasebeing treated.

In case of a medicament intended for the treatment of chronicobstructive pulmonary disease (COPD) it will preferably be presented inthe form suitable for inhaled administration via the inhalation route.

As defined above, X− may be any physiologically acceptable counterion.Thus, salts of the formula I may be derived from any physiologicallyacceptable acid, both organic and inorganic. Suitable salts withinorganic acids are for example chloride, bromide, iodide and carbonate;suitable salts with organic acids may be salts with mono-, di- andtricarboxylic acids, for example acetate, benzoate, salicylate,hydroxyacetate, lactate, malonate and citrate. Preferred salts arechlorides, benzoates, salicylates, acetates, citrates and lactates;especially advantageous are chlorides.

Specific compounds of the formula (I) are 1-methylnicotinamide salts(MNA⁺), 1-methyl-3-acetylpyridinium salts (MAP⁺) and1-methyl-N′-(hydroxymethyl)nicotinamide salts (MNAF⁺).

The invention in the second aspect provides a method of treatment and/orprevention of conditions or diseases associated with dysfunction ofvascular endothelium, oxidative stress, and/or insufficient productionof endothelial PGI₂ (associated or not with hypercholesterolemia,hypertriglyceridemia or low HDL level), in particular such as discussedabove, comprising administration to a subject in a need of suchtreatment a therapeutically effective amount of a quaternary pyridiniumsalt of formula I as defined above

In one embodiment of the method of treatment according to the inventionsaid condition or the disease is atherosclerosis of vascular bed of anykind, including chronic coronary disease, ischemic cerebrovascularepisode or artherosclerosis of the extremities, includingthrombo-angigitis obliterans.

In another embodiment of the method of treatment according to theinvention the said condition or disease is acute cardiovascular eventassociated with atherosclerosis, in particular sudden cardiac death,acute coronary syndrome (including unstable coronary artery disease,myocardial infarct), the necessity of coronary angioplasty (PCI),coronary-aortal by-pass surgery (CABG), ischemic stroke, or peripheralcirculation revascularization.

In yet another embodiment of the method of treatment according to theinvention said condition or disease is selected from the group of riskfactors for atherosclerosis, comprising the following:hypercholesterolemia, arterial hypertension, smoking,hyperhomocysteinaemia, insulin resistance, diabetes, menopause, aging,obesity, mental stress, infections, inflammatory states, includingperiodontal diseases, reumathoid arthritis, allograft vasculopathy ornitrate tolerance.

In yet another embodiment of the method of treatment according to theinvention said condition or the disease is dyslipidemia, in particularhypercholesterolemia or hypertriglyceridemia, in particular associatedwith low plasma level of HDL.

In yet another embodiment of the method of treatment according to theinvention said condition or disease is thrombosis that is not relateddirectly with atherosclerosis, in particular thrombosis associated withimplantation of metallic vascular prostheses (stents), coronary-aortalby-pass surgery(CABG), any type of surgery with extracorporealcirculation, hemodialysis, venous thrombo-embolic disease.

In a further embodiment of the method of treatment according to theinvention said condition or disease is selected from the followinggroup: chronic heart failure, pulmonary hypertension, microvasculardiabetic complications, like diabetic retinopathy and nephropathy,diabetic neuropathy, nephrotic syndrome, chronic renal failure, adultsrespiratory distress syndrome (ARDS), cystic fibrosis, chronicobstructive pulmonary disease (COPD), preeclampsia/eclampsia, erectiledysfunction, Stein-Leventhal syndrome, sleep apnea, systemic lupuserythematosus, sickle cell anemia, non-specific inflammatory boweldiseases, gastric or duodenal ulcers, glaucoma, chronic liver disease,primary amyloidosis, neurodegenerative diseases, in particularneurodegenerative disease selected from vascular dementia, Alzheimer'sdisease and Parkinson's disease.

In a further preferred embodiment said condition or disease is chronicliver disease, in particular chronic viral hepatitis.

In a further preferred embodiment said condition or disease is chronicobstructive pulmonary disease (COPD).

Also advantageous is the use of the compounds of formula I in the methodof prophylaxis or treatment of gastric or duodenal ulcer.

Quaternary pyridinium salts of formula I may be administered alone or incombination with other cardiovascular agent.

Pyridinium salts of formula I may be administered in particular orally,in the form of conventional oral preparations, such as tablets,capsules, oral solutions/suspensions in pharmaceutically acceptableliquid carrier. Said formulations may be prepared using conventionalmethods known in the art and include conventional pharmaceuticalexcipients and carriers.

Pyridinium salts of formula I may be also administered parenterally, inthe form of injections, including subcutaneous and intravenousinjections and infusions.

Other contemplated routes of administration are by inhalation,intranasally and rectally.

In any case the route of administration will of course depend on theparticular disease being treated.

For example, in case of a treatment of chronic obstructive pulmonarydisease (COPD) pyridiniurn salts it will preferably be administered inthe form suitable for inhaled administration via the inhalation route.

Daily dose of the pyridinium salts of the formula I may be in the rangeof 10 to 1000 mg and may be administered in single or divided doses.

The present invention relates also to a method for enhancing aprostacyclin levels in mammals, which comprises oral administration ofeffective amount of quaternary pyridinium salt of formula I as definedabove.

The present invention provides also quaternary pyridinium salts offormula I as defined above, for use in oral diet supplementation.Pyridinium salts of formula I, when used as an oral diet supplement,enhance the prostacyclin level, thereby acting as vasoprotective agents.

The present invention provides further a use of pyridinium salts of theformula I as defined above, for the preparation of nutritionalpreparation for vascular protection in mammals in states or diseasesassociated with dysfunction of vascular endothelium, oxidative stress,and/or insufficient production of endothelial prostacyclin (PGI₂), inparticular associated with hypercholesterolemia, hypertriglyceridemia orlow HDL level.

Said condition or disease wherein nutritional preparation may beadministered is atherosclerosis, especially in patients with chroniccoronary disease, ischemic cerebrovascular episode or artherosclerosisof the extremities, including thromboangigitis obliterans.

Said condition or disease wherein nutritional preparation may beadministered may be also selected from the group of risk factors foratherosclerosis, comprising the following: hypercholesterolemia,arterial hypertension, smoking, hyperhomocysteinaemia, insulinresistance, diabetes, menopause, aging, obesity, mental stress,infections, inflammatory states, including periodontal diseases,reumathoid arthritis, allograft vasculopathy or nitrate tolerance.

Said condition or disease wherein nutritional preparation may beadministered is dyslipidemia, in particular hypercholesterolemia,hypertriglyceridemia in particular associated with low plasma level ofHDL.

Said condition or disease wherein nutritional preparation may beadministered may be also thrombosis that is not related directly withatherosclerosis, in particular thrombosis associated with implantationof metallic vascular prostheses (stents), coronary-aortal by-passsurgery (CABG), any type of surgery with extracorporeal circulation,hemodialysis, venous thrombo-embolic disease.

Specific pyridinium salts for use in diet supplementation and/or asnutritional preparation are compounds of the formula (I), wherein R isCH₃ group.

Specific pyridinium salts for use in diet supplementation and/or asnutritional preparation are compounds of the formula (I), wherein R isNH₂ group.

Specific pyridinium salts for use in diet supplementation and/or asnutritional preparation are compounds of the formula (I), wherein R isN(H)CH₂OH group.

Dietary supplements and nutritional preparations may have the formsuitable for oral ingestion, such as tablets, capsules, solutions andsuspensions for drinking, and similar, conventional and known inpharmaceutical art and prepared according to techniques known in the artwith the use of conventional excipients and carriers.

Advantageously, dietary supplement or nutritional preparation mayincorporate at least 5% by weight of the pyridinium salt of the formulaI.

The invention is further illustrated by the following Examples, whichshow pharmacological activity of pyridinium salts.

EXAMPLE I Thrombolytic Activity

Thrombolytic activity of quateixiary pyridinium salts was assessed usingoriginal method of Gryglewski et al., (Gryglewski R J, Korbut R,Ocetkiewicz A, Stachura J. In vivo method for quantitation foranti-platelet potency of drugs. Naunyn Schmiedebergs Arch Pharmacol1978;302:25-30), the scheme of which was shown on FIG. 1.

Wistar rats of body weight 300-350 g were anaesthetised (thiopental 30mg kg⁻¹ i.p.) and heparinised (unfractionated heparin 800 i.u. kg⁻¹i.v.). Arterial blood pressure was measured from cannulated rightcarotid artery, and extracorporal circulation was established betweenleft carotid artery and left jugular vein. A collagen strip from rabbittendon of Achilles was superfused with arterial blood in extracorporalcirculation at a rate of 1.5 ml min⁻¹ and its weight was continuouslymonitored by an auxotonic Harvard transducer.

During the initial 20-30 mM of superfusion the strip was gaining inweight by 80-120 mg because of deposition of platelet-rich thrombi andthen in control conditions stayed unchanged during next 3-5 hrs.Thrombolytic response was detected by a fall in weigth of a thrombi.Arterial blood pressure was also monitored, so this model enabled theanalysis of thrombolytic and hypotensive action of a compound (FIG. 1).

The analysis of the thrombolytic response in this experimental set-upwas complemented by the assay of 6-keto-PGF_(1α), TXB₂ and PGE₂ inarterial blood plasma. For this purpose blood samples (500 μl) werecollected in Eppendorff tubes with indomethacin to yield its finalconcentration of 10 μM, and EDTA to yield the final concentration of 1mM. Then, the blood samples were spinned for 5 min at 2.000×g. Plasmasamples were stored at −70° C. The prostanoids were assayed using theenzyme immunoassay kits (Cayman Chemical Co, Ann Arbor, Mich.).

Intravenous administration of MNA⁺ (3-30 mg/kg) produced aconcentration-dependent thrombolysis in Wistar rats with extracorporalcirculation. A maximum response was observed at the MNA⁺ dose of 30mg/kg. Single injection of MNA⁺ at a dose of 30 mg/kg induced along-lasting thrombolytic response at the level of 42±4% and remained atapproximately the same level for 2-3 hours of the observation period. Incontrast to MNA⁺, nicotinamide, nicotinic acid, trigonelline and 2-PYR(endogenous metabolite of MNA⁺), each of them at 30 mg/kg, failed toinduce a significant thrombolytic response. Nicotinamide and nicotinicacid-induced responses were transient (less then 15-20 minutes) and attheir maximum amounted merely to 9±0.6%, 5±0.9%, respectively).Trigonelline did not produce any thrombolytic response and response to2-PYR was also very weak (<10%) and transient (<15 min). The potency andduration of thrombolytic responses to MNA⁺, nicotinamide and nicotinicacid correlated with a pattern of 6-keto-PGF_(1α) release to arterialplasma induced by these compounds. MNA⁺ (30 mg/kg) induced a substantialincrease in levels of 6-keto-PGF_(1α) as early as 15 minutes after druginjection (from 104±7 to 460±58 pg/ml) which than reached its plateau ofaround 400 pg/ml for at least one hour. On the other hand neither TXB2nor PGE₂ levels changed significantly in response to MNA⁺. Sluggish risein TXB₂ levels was time-dependent and observed also after salineinjection. Levels of 6-keto-PGF_(1α) did not increase after injection ofnicotinamide or after injection of nicotinic acid (30 mg/kg).

In the presence of indomethacin (5 mg/kg) thrombolytic response to MNA⁺was abrogated, similarly as MNA-induced release of 6-keto-PGF_(1α).Importantly, thrombolysis induced by MNA⁺ (30 mg/kg) was not associatedwith a fall in arterial blood pressure. Collagen-induced aggregation inplatelet-rich plasma in vitro was not affected by MNA⁺ up toconcentration of 10 mM, this excluding the possibility that dissipationof plate-let-rich thrombi in vivo was due to the direct effect of MNA⁺on platelets. Furthermore MNA⁺ (1 mM) did not inhibit latex-inducedactivation of neutrophils, this suggesting a possible selectivity ofMNA⁺ towards endothelium.

FIG. 2 shows thrombolytic response in vivo induced by MNA⁺, and FIG. 3shows a concomitant increase in level of 6-keto-PGF_(1α) (stable PGI₂metabolite) in blood. Nicotinamide, nicotinic acid (FIG. 4),trigonelline and 2-PYR (FIG. 5) did not display a significantthrombolytic activity. However, MAP⁺ (30 mg/kg) and MNAF⁺ (30 mg/kg)each of them induced a thrombolytic response (FIG. 6 and FIG. 8) thatwas comparable to that induced by 30 mg/kg of MNA⁺. MAP⁺—inducedthrombolysis was associated with the release of PGI₂ (FIG. 7), similarlyas in the case of MNA⁺-induced thrombolysis. MNAF (30-300 mg/kg) did notcause hypotension. As shown on FIG. 9 and FIG. 10, MNA⁺ did not inhibitcollagen-induced aggregation of platelets and latex-induced activationof neutrophils. The former response depends on COX1-TXA₂ and wasabrogated by aspirin, whereas the latter one depends on NADPH oxidaseand was abrogated by DPI or apocynin.

EXAMPLE 2 An Ant-Athrogenic Effect In Vivo in Patients

The anti-atherogenic effect of MNA⁺ was investigated in 15 dyslipidemicpatients.

The enrollment criteria were: high level of TG (≧250 mg/dl) and lowlevel of HDL (≦35 mg/dl for male, ≦45 mg/dl for female). The mean age ofthe patients was 61.4 (range 45-81 years).

The patients were treated with MNA⁺ for 2 weeks. The MNA⁺ wasadministered orally, three times a day, one capsule (30 mg MNA⁺) aftermeal.

The plasma levels of TC, TG, HDL were measured at baseline and after 2weeks of therapy. The level of LDL was measured in those cases, where itwas possible (due to high TG level).

It has been found that MNA⁺ reduced the TC (267.0 vs. 225.1 mg/dl)(−15.7%) and TG (472.6 vs 249.9 mg/dl) (−47.1%) levels between thebaseline and 2 weeks measurements. The increase of the HDL (39.2 vs.53.4 mg/dl) (36.2%) level was also observed after 2 weeks therapy. Thesignificant reduction of TG/HDL ratio (13.9 vs. 5.8) was observed.

1-56. (canceled)
 57. A method for treatment of dyslipidemia,hypercholesterolemia, hypertriglyceridemia or hypertriglyceridemiaassociated with low plasma level of HDL in a subject in need thereof,said method comprising administering to said subject a therapeuticallyeffective amount of a quaternary pyridinium salt of formula I:

wherein R is NH₂, CH₃ or N(H)CH₂OH, and X is a pharmaceuticallyacceptable counterion.
 58. The method in accordance with claim 57,wherein said administering lowers said subject's plasma triglyceridelevel.
 59. The method in accordance with claim 57, wherein saidadministering raises said subject's plasma HDL level.
 60. The method inaccordance with claim 57, wherein said quaternary pyridinium acceptablesalt is administered orally.
 61. The method in accordance with claim 57,wherein said quaternary pyridinium acceptable salt is administeredparenterally.
 62. The method in accordance with claim 57, wherein saidquaternary pyridinium acceptable salt is in the form of a compositionwith a pharmaceutically acceptable carrier.
 63. The method of claim 57,for the treatment of hypertriglyceridemia associated with a low plasmalevel of HDL.
 64. The method of claim 57, wherein said quaternarypyridinium acceptable salt is a 1-methyl-3-acetylpyridinium salt. 65.The method of claim 57, wherein said quaternary pyridinium acceptablesalt is a 1-methyl-N′-(hydroxymethyl) nicotinamide salt.
 66. The methodof claim 57, wherein said quaternary pyridinium acceptable salt is a1-methylnicotinamide salt.
 67. A method for treatment of a diseaseassociated with dysfunction of vascular endothelium, oxidative stress,or insufficient production of endothelial prostacyclin in a subject inneed thereof, said method comprises administering to said subject aneffective amount of a quaternary pyridinium salt of formula I:

wherein R is NH₂, CH₃ or N(H)CH₂OH, and X is a pharmaceuticallyacceptable counterion.
 68. The method of claim 67, wherein said diseaseis selected from the group consisting of: chronic heart failure,pulmonary hypertension, diabetic retinopathy, diabetic nephropathy,nephrotic syndrome, chronic renal failure, adults respiratory distresssyndrome (ARDS), cystic fibrosis, chronic obstructive pulmonary disease(COPD), preeclampsia, eclampsia, erectile dysfunction, Stein-Leventhalsyndrome, sleep apnea, systemic lupus erythematosus, sickle cell anemia,non-specific inflammatory bowel diseases, gastric ulcers, duodenalulcers, glaucoma, chronic liver diseases, primary amyloidosis, vasculardementia, Alzheimer's disease and Parkinson's disease.
 69. The method ofclaim 68, wherein said disease is duodenal ulcer.
 70. The method ofclaim 68, wherein said disease is chronic obstructive pulmonary disease(COPD).
 71. The method of claim 67, wherein said quaternary pyridiniumsalt is administered orally.
 72. The method of claim 67, wherein saidquaternary pyridinium salt is administered via the inhalation route. 73.The method of claim 67, wherein said quaternary pyridinium salt isadministered parenterally.
 74. The method of claim 67, wherein saidquaternary pyridinium salt is administered in the form of a compositionwith a pharmaceutically acceptable carrier.
 75. The method of claim 67,wherein said mammal has an insufficient production of endothelialprostacyclin.
 76. The method of claim 75, wherein said insufficientproduction of endothelial prostacyclin is age-related.
 77. The method ofclaim 67, wherein said quaternary pyridinium salt is a1-methylnicotinamide salt.
 78. The method of claim 67, wherein saidquaternary pyridinium salt is a 1-methyl-3-acetylpyridinium salt. 79.The method of claim 67, wherein said quaternary pyridinium salt is a1-methyl-N′-(hydroxymethyl) nicotinamide salt.
 80. The method of claim67, wherein X is a counterion acceptable for consumption.
 81. The methodof claim 80, wherein said quaternary pyridinium salt is in the form of anutritional preparation or an oral dietary supplement.
 82. The method ofclaim 80, wherein said nutritional preparation or said oral dietarysupplement contains at least 5% by weight of said quaternary pyridiniumsalt.
 83. The method of claim 67, wherein from about 10 mg to about 1000mg of said quaternary pyridinium salt is administered daily, in singleor divided doses.